Therapeutic agent for fibroid lung

ABSTRACT

Disclosed are: a substance transfer carrier to an extracellular matrix-producing cell in the lung, which comprises a retinoid; a therapeutic agent for fibroid lung, which utilized the carrier; and a preparation kit of the therapeutic agent.

BACKGROUND OF THE INVENTION

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

1. Sequence Listing

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledKUZU1_(—)002C2.TXT, created Apr. 19, 2013, which is 1.87 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

2. Field of the Invention

The present invention relates to a substance delivery carrier targetedat extracellular matrix-producing cells in the lung, and a therapeuticagent for pulmonary fibrosis and a method for treating pulmonaryfibrosis utilizing the carrier.

3. Description of the Related Art

Pulmonary fibrosis is a disease characterized by diffuse fibroplasia ofthe alveolar walls, and its main symptoms include dry cough and dyspneaon exertion. In a restricted sense, it refers to end-stage diseasestates of interstitial pneumonia; while in a broad sense, it means aco-existing state of pulmonary fibrosis in a restricted sense withinterstitial pneumonia. Any interstitial pneumonia can cause pulmonaryfibrosis. Interstitial pneumonia is a generic term for the diseases thatinduce inflammation in interstices of the lung (including alveolarseptum in a restricted sense, and intralobular interstice and thevicinity of pleural membrane in a broad sense); it includes thoseinduced by a specific cause such as infection, collagen disease,radiation, drug, and dust, and those without any known cause, i.e.,idiopathic interstitial pneumonia. Idiopathic interstitial pneumonia isfurther classified as follows based on the findings of video-assistedthoracoscopic surgery (VATS) and high-resolution computer tomography(HRCT): idiopathic pulmonary fibrosis (IPF), nonspecific interstitialpneumonia (NSIP), acute interstitial pneumonia (AIP), cryptogenicorganizing pneumonia (COP), respiratory bronchiolitis-associatedinterstitial lung disease (RB-ILD), desquamative interstitial pneumonia(DIP), lymphoid interstitial pneumonia (LIP), etc. Many of theinterstitial pneumonia with specified causes are cured by elimination ofthe causes and administration of anti-inflammatory agents such assteroid drugs. However, regarding idiopathic interstitial pneumonia,there is no radical treatment method to date, and only treatments suchas administration of steroid drugs, azathioprine and cyclophosphamideduring exacerbation of symptoms, and oxygen therapy during developmentof hypoxemia are performed; accordingly, there are many dead cases inwhich idiopathic interstitial pneumonia progresses into pulmonaryfibrosis. Therefore, the average survival period after establishment ofdiagnosis of idiopathic interstitial pneumonia is as short as 2.5-5years, and this disease is designated as one of the specific diseases inJapan.

Under such circumstances, much research effort has been made to thedevelopment of therapeutic agents for pulmonary fibrosis. As a result,pharmaceutical agents such as colchicine, D-penicillamine, pirfenidone(5-methyl-1-phenyl-2-[1M-pyridone), interferon-β1a, relaxin, lovastatin,beractant, N-acetylcysteine, keratinocyte growth factor, captopril (NonPatent Literature 1), hepatocyte growth factor (Patent Literature 1),Rho-kinase inhibitor (Patent Literature 2), thrombomodulin-like protein(Patent Literature 3), bilirubin (Patent Literature 4), PPARγ(peroxisome proliferator-activated receptor gamma) activator (PatentLiterature 5), imatinib (Patent Literature 6), interferon-γ (PatentLiterature 7) have been reported to show some effectiveness in animalmodels of pulmonary fibrosis or clinical trials. However, none of theseagents is not yet satisfactory, and further development of thetherapeutic agents for pulmonary fibrosis has been awaited.

CITATION LIST

Patent Literature 1: JP A No. 8-268906

Patent Literature 2: WO 00/57913

Patent Literature 3: JP A No. 2002-371006

Patent Literature 4: JP A No. 2003-119138

Patent Literature 5: JP A No. 2005-513031

Patent Literature 6: JP A No. 2005-531628

Patent Literature 7: JP A No. 2006-502153

Patent Literature 8: WO 2006/068232

Non Patent Literature 1: Ann Intern Med. 2001; 134(2): 136-51

SUMMARY OF INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a carrier that candeliver a substance such as drugs specifically to extracellularmatrix-producing cells in the lung, as well as a therapeutic agent forpulmonary fibrosis and a method for treating pulmonary fibrosisutilizing said carrier.

Means for Solving the Problems

The inventors of the present invention have searched for noveltherapeutic agents for pulmonary fibrosis, and found that theadministration of a composition in which a carrier comprising a retinoidcarries an inhibitor for the production of extracellular matrix caneffectively treat pulmonary fibrosis; then the inventors have completedthis invention.

While it has been known that a carrier comprising vitamin A can delivera drug to stellate cells that store vitamin A (refer to PatentLiterature 8), the relationship with pulmonary fibrosis has beencompletely unknown to date.

Namely, the present invention relates to the following:

(1) A substance delivery carrier to an extracellularmatrix-producingcell in the lung, comprising a retinoid as a targeting agent.

(2) The carrier according to the above (1), wherein the retinoidderivative comprises retinol.

(3) The carrier according to the above (1) or (2), wherein the retinoidcontent is 0.2-20 wt % of the entire carrier.

(4) The carrier according to any one of the above (1) to (3), whereinthe carrier has a form of liposome, and the molar ratio of the retinoidto the lipid contained in the liposome is 8:1-1:4.

(5) A composition for treating pulmonary fibrosis, comprising thecarrier according to any one of the above (1) to (4) and a drug thatcontrols the activity or growth of extracellular matrix-producing cellsin the lung.

(6) The composition according to the above (5), wherein the drug thatcontrols the activity or growth of extracellular matrix-producing cellsin the lung is selected from the group consisting of an agent forinhibiting activity or production of a bioactive substance selected fromthe group consisting of gelatinase A, gelatinase B and angiotensinogen,an inhibitor of cell activity, a growth inhibitor, an apoptosis-inducingagent, as well as an siRNA (small interfering RNA), a ribozyme, an anti-sense nucleic acid, and a DNA/RNA chimeric polynucleotide which targetat least one of extracellular matrix constituent molecules or moleculesinvolved in the production or secretion of said extracellular matrixconstituent molecules, and a vector that expresses said siRNA, ribozyme,anti-sense nucleic acid, and DNA/RNA chimeric polynucleotide.

(7) The composition according to the above (6), wherein the moleculeinvolved in the production or secretion of the extracellular matrixconstituent molecules is HSP (heat shock protein) 47.

(8) The composition according to any one of the above (5) to (7),wherein the drug and the carrier are mixed at a place of medicaltreatment or in its vicinity.

(9) A kit for preparing the composition according to any one of theabove (5) to (8), wherein the kit comprises one or more containerscomprising singly or in combination a drug for inhibiting activity orgrowth of extracellular matrix-producing cells in the lung, a retinoid,and if necessary, a carrier-constituent substance other than retinoid.

Effects of the Invention

While the exact mechanism of action of the composition for treatingpulmonary fibrosis of the present invention has not yet been completelyclarified, the mechanism is considered as follows: with the composition,retinoid functions as a targeting agent to extracellularmatrix-producing cells in the lung such as fibroblasts andmyofibroblasts, and the retinoid delivers an active ingredient such aspharmaceutical agents that control activity or growth of extracellularmatrix-producing cells in the lung to such cells, thereby exhibiting theeffect against pulmonary fibrosis.

Accordingly, since active ingredients can be efficiently delivered toaction sites, and further to targeted cells by using the carrier of thepresent invention, the treatment, suppression of progression, andprevention of onset of pulmonary fibrosis, in particular idiopathicinterstitial pneumonia the treatment of which has been difficult to dateare made possible; thus, the inventive carrier significantly contributesto the human medicine and veterinary medicine.

Moreover, the carrier of the present invention can be combined with anypharmaceutical drugs (for example, existing therapeutic agents forpulmonary fibrosis) to increase their action efficiency; therefore, itis also advantageous as its application range in terms of formulation isbroad, facilitating the production of effective therapeutic agents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing induction of pulmonary fibrosis inrats and drug-administration schedule.

FIG. 2 is a graph showing the total number of cells in BAL liquid on day21 after administration of bleomycin. “Control” means normal ratswithout administration of bleomycin.

FIG. 3 is a graph showing the amount of hydroxyproline (HP) In the lungon day 21 after administration of bleomycin. “Control” means normal ratswithout administration of bleomycin.

FIG. 4 shows photographs of HE-stained lung tissues on day 21 afteradministration of bleomycin.

FIG. 5 shows photographs of Azan-stained lung tissues on day 21 afteradministration of bleomycin.

FIG. 6 shows photographs showing distribution of αSMA-positive cells inthe lung tissues on day 21 after administration of bleomycin.

DESCRIPTION OF EMBODIMENTS

In the present invention, the extracellular matrix-producing cells inthe lung are not particularly limited as long as they are cells presentin the lung and having a capability of producing extracellular matrix,and they include, for example, fibroblasts and myofibroblasts present inthe lung. Fibroblasts present in the lung include, for example, vascularadventitial fibroblasts and bronchiolar adventitial fibroblasts, etc.Myofibroblasts present in the lung may include not only those derivedfrom such fibroblasts present in the lung, but also those derived fromfibroblasts in the circulating blood and those transformed fromendothelial cells by endothelial mesenchymal transdifferentiation.Myofibroblasts are characterized by expression of α-smooth muscle actin(α-SMA). The myofibroblasts in the present invention are thoseidentified, e.g., by immunostaining using detectably-labeled anti-α-SMAantibodies. In addition, while fibroblasts express vimentin that ischaracteristic to mesenchymal cells, they do not express α-SMA;therefore, fibroblasts can be identified by double-staining withvimentin and α-SMA.

The retinoid of the present invention is not particularly limited aslong as it promotes delivery of a substance to extracellularmatrix-producing cells in the lung, and examples thereof includeretinoid derivatives such as retinol (vitamin A), etretinate, tretinoin,isotretinoin, adapalene, acitretine, tazarotene, and retinol palmitate,as well as vitamin A analogues such as fenretinide (4-HPR,4-hydroxyphenylretinamide) and bexarotene.

The retinoid of the present invention is that which promotes specificdelivery of a substance to extracellular matrix-producing cells in thelung. The mechanism of the promotion of substance delivery by retinoidhas not yet been completely clarified; however, the following mechanismis considered: for example, a retinoid which has specifically bound to aretinol-binding protein (RBP) is taken into an extracellularmatrix-producing cell in the lung through a certain receptor present onthe surface of this cell.

A retinoid is a member of the class of compounds having a skeleton inwhich four isoprenoid units are bonded in a head-to-tail manner (see G.P. Moss, “Biochemical Nomenclature and Related Documents,” 2nd Ed.Portland Press, pp. 247-251 (1992)). Vitamin A is a generic descriptorfor a retinoid that qualitatively shows the biological activity ofretinol. Retinoid that can be used in the present invention are notparticularly limited, and examples thereof include retinoid derivativessuch as retinol, retinal, retinoic acid, an ester of retinol and a fattyacid, an ester of an aliphatic alcohol and retinoic acid, etretinate,tretinoin, isotretinoin, adapalene, acitretine, tazarotene and retinolpalmitate, and vitamin A analogues such as fenretinide (4-HPR) andbexarotene.

Of these, retinol, retinal, retinoic acid, an ester of retinol and afatty acid (such as retinyl acetate, retinyl palmitate, retinyl stearateand retinyl laurate) and an ester of an aliphatic alcohol and retinoicacid (such as ethyl retinoate) are preferable from the viewpoint ofefficiency of specific delivery of a substance to extracellularmatrix-producing cells in the lung.

All retinoid isomers, such as cis-trans, are included in the scope ofthe present invention. The retinoid may be substituted with one or moresubstituents. The retinoid in the present invention includes a retinoidin an isolated state as well as in a solution or mixture state with amedium that can dissolve or retain the retinoid.

The carrier of the present invention may be formed from the retinoid onits own or may be formed by making the retinoid bind to or enclosed in acarrier constituent component other than the retinoid. Therefore, thecarrier of the present invention may comprise a carrier constituentcomponent other than the retinoid. Such a component is not particularlylimited, and any component known in the medicinal and pharmaceuticalfields may be used, but those that can enclose retinoid or can bindthereto are preferable.

Examples of such a component include a lipid, for example, aphospholipid such as glycerophospholipid, a sphingolipid such assphingomyelin, a sterol such as cholesterol, a vegetable oil such assoybean oil or poppy seed oil, a mineral oil, and a lecithin such asegg-yolk lecithin, but the examples are not limited thereto. Among them,those that can form a liposome are preferable, for example, a naturalphospholipid such as lecithin, a semisynthetic phospholipid such asdimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), or distearoylphosphatidylcholine (DSPC), anddioleylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine(DLPC), and cholesterol.

A particularly preferred component is a component that can avoid captureby the reticuloendothelial system, and examples thereof include cationiclipids such as N-(α-trimethylammonioacetyl)-didodecyl-D-glutamatechloride (TMAG),N,N′,N″,N′″-tetramethyl-N,N′,N″,N′″-tetrapalmitylspermine (TMTPS),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),dioctadecyldimethylammonium chloride (DODAC), didodecylammonium bromide(DDAB) 1,2-dioleyloxy-3-trimethylammoniopropane (DOTAP),3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol),1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE), andO,O′-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride(DC-6-14).

The binding of the retinoid to the carrier of the present invention orthe enclosing of it therein is also possible by binding or enclosing theretinoid to or in a constituent component, other than the retinoid, ofthe carrier by a chemical and/or physical method. Alternatively, thebinding or enclosing the retinoid to or in the carrier of the presentinvention can also be carried out by mixing the retinoid and aconstituent component, other than the retinoid, of the carrier whenpreparing the carrier. The amount of retinoid bound to or enclosed inthe carrier of the present invention may be, as a weight ratio in thecarrier constituent components, 0.01% to 100%, preferably 0.2% to 20%,and more preferably 1% to 5%. The binding or enclosing of retinoid to orin the carrier may be performed before a drug, etc. is carried on thecarrier, may be performed by simultaneously mixing the carrier, aretinoid derivative and a drug, etc., or may be performed by mixing aretinoid derivative with a carrier on which a drug, etc. is alreadycarried. Therefore, the present invention also relates to a process forproducing a formulation specific to extracellular matrix-producing cellsin the lung, the process including a step of binding a retinoid to anyexisting drug binding carrier or drug encapsulating carrier, forexample, a liposomal formulation such as DaunoXome®, Doxil, Caelyx®, orMyocet®.

The form of the carrier of the present invention may be any form as longas a desired substance or matter can be transported to a targetextracellular matrix-producing cell in the lung, and although notlimited thereto, examples thereof include a macromolecular micelle, aliposome, an emulsion, microspheres, and nanospheres. In the presentinvention, from the viewpoint of high delivery efficiency, wideselection of substances to be delivered, and ease of making aformulation, etc., a liposomal form is preferable among the forms, and acationic liposome that includes a cationic lipid is particularlypreferable. In the case that the carrier is a liposomal form, the molarratio of the retinoid to liposome constituent components other than theretinoid is, considering the efficiency of retinoid's binding to orenclosing in the carrier, preferably 8:1 to 1:4, more preferably 4:1 to1:2, yet more preferably 3:1 to 1:1, and particularly preferably 2:1.

The carrier of the present invention may contain a substance to betransported within its interior, maybe attached to the exterior of asubstance to be transported, or may be mixed with a substance to betransported, as long as retinoid contained therein is present in such aform that it can function as a targeting agent. “Function as a targetingagent” referred to here means that the carrier containing retinoidreaches and/or is taken up by the target cell, i.e., extracellularmatrix-producing cells in the lung, more rapidly and/or in a largerquantity than with a carrier not containing retinoid, and this mayeasily be confirmed by, for example, adding a labeled orlabel-containing carrier to a culture of target cells, and analyzing thesites where the label is present after a predetermined period of time.Structurally, this requirement can be satisfied, for example, ifretinoid is at least partially exposed to the exterior of a formulationcontaining the carrier at the latest by the time it reaches the targetcell. Whether or not the retinoid is exposed at the exterior of aformulation can be evaluated by contacting the formulation to asubstance that specifically binds to retinoid, such as a retinol-bindingprotein (RBP), and evaluating the binding to the formulation.

The substance or matter that is delivered by the present carrier is notparticularly limited, and it preferably has a size such that it canphysically move within the body of a living being from an administrationsite to a lesion site where a target cell is present. Therefore, thecarrier of the present invention can transport not only a substance suchas an atom, a molecule, a compound, a protein, or a nucleic acid, butalso a matter such as a vector, a virus particle, a cell, adrug-releasing system formed from one or more elements, or amicromachine. The above substance or matter preferably has the propertyof having some influence on a target cell, and examples thereof includethose that label a target cell and those that control (e.g. increase orsuppress) the activity or growth of a target cell.

Therefore, in one embodiment of the present invention, the substancethat the carrier delivers is “a drug controlling the activity or growthof an extracellular matrix-producing cell in the lung”. The activity ofan extracellular matrix-producing cell in the lung referred to hereindicates various activities such as secretion, uptake, migration, etc.exhibited by an extracellular matrix-producing cell in the lung, and inthe present invention among them it typically means, in particular,activities involved in the onset, progression, and/or recurrence ofpulmonary fibrosis. Examples of such activities include, but are notlimited to, production/secretion of a bioactive substance such asgelatinase A and gelatinase B (MMP (matrix metalloproteinase) 2 and MMP9, respectively) andangiotensinogen, etc., and an extracellular matrixcomponent such as collagen, proteoglycan, tenascin, fibronectin,thrombospondin, osteopontin, osteonectin, and elastin.

Therefore, the drug controlling the activity or growth of anextracellular matrix-producing cell in the lung may be any drug thatdirectly or indirectly suppresses the physical, chemical, and/orphysiological actions, etc. of said cell related to the onset,progression, and/or recurrence of pulmonary fibrosis, and while notbeing limited thereto, it includes the following: drugs inhibiting theactivity or production of the above bioactive substances, MMP inhibitorssuch as batimastat, and antibodies and antibody fragments thatneutralize the above bioactive substances, and substances that suppressexpression of the above bioactive substances, such as an siRNA, aribozyme, an antisense nucleic acid (including RNA, DNA, PNA (peptidenucleic acid), or a composite thereof), and substances that have adominant negative effect such as a dominant negative mutant, or a vectorexpressing the same, drugs suppressing the production and secretion ofthe above extracellular matrix component, etc., for example, substancesthat suppress expression of the extracellular matrix component, such asan siRNA, a ribozyme, an antisense nucleic acid (including RNA, DNA,PNA, or a composite thereof), and substances that have a dominantnegative effect such as a dominant negative mutant, or a vectorexpressing the same, inhibitors of cell activity such as a sodiumchannel blocker, cell-growth inhibitors, for example alkylating agents(such as ifosfamide, nimustine, cyclophosphamide, dacarbazine,melphalan, and ranimustine), antitumor antibiotics (such as idarubicin,epirubicin, daunorubicin, doxorubicin, pirarubicin, bleomycin,peplomycin, mitoxantrone, and mitomycin C), antimetabolites (such asgemcitabine, enocitabine, cytarabine, tegafur/uracil, ategafur/gimeracil/oteracil potassium mixture, doxifluridine,hydroxycarbamide, fluorouracil, methotrexate, and mercaptopurine),alkaloids such as etoposide, irinotecan, vinorelbine, docetaxel hydrate,paclitaxel, vincristine, vindesine, and vinblastine, and platinumcomplexes such as carboplatin, cisplatin, and nedaplatin, and apoptosisinducers such as compound 861, gliotoxin, lovastatin, and Beractant.Furthermore, the “drug controlling the activity or growth of anextracellular matrix-producing cell in the lung” in the presentinvention may be any drug that directly or indirectly promotes thephysical, chemical, and/or physiological actions, etc. of anextracellular matrix-producing cell In the lung directly or indirectlyrelated to the suppression of onset, progression, and/or recurrence ofpulmonary fibrosis.

The substance delivered by the carrier of the invention include, withoutlimitation, drugs other than those mentioned above and which suppressthe onset, progression, and/or recurrence of pulmonary fibrosis, andexamples include, but are not limited to, colchicine, D-penicillamine,pirfenidone (5-methyl-l-phenyl-2-[1H]-pyridone), interferon-β1a,relaxin, N-acetylcysteine, keratinocyte growth factor, captopril,hepatocyte growth factor, Rho kinase inhibitor, thrombomodulin-likeprotein, bilirubin, PPARγ activator, imatinib, interferon-γ, and TGFβreceptor kinase inhibitor.

The substance or matter delivered by the carrier of the presentinvention mayor may not be labeled. Labeling enables monitoring of thesuccess or failure of transport, increases and decreasestesting/research level. A label may be selected from any label known toa person skilled in the art such as, for example, any radioisotope,magnetic material, substance that binds to a labeling substancematrix-producing cell in the lung” or “for the delivery to anextracellular matrix-producing cell in the lung” means that it issuitable to use to extracellular matrix-producing cells as a target, andthis includes it being possible to deliver a substance to this cell,more rapidly, efficiently, and/or in a larger quantity than to othercells, for example, normal cells. For example, the carrier of thepresent invention can deliver a substance to an extracellularmatrix-producing cell in the lung at a rate and/or efficiency of 1.1times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more,2 times or more, or even 3 times or more compared with other cells.

The present invention also relates to a composition for controlling theactivity or growth of an extracellular matrix-producing cell in thelung, or for treating pulmonary fibrosis, that comprises the carrier,and the drug controlling the activity or growth of an extracellularmatrix-producing cell in the lung, and to a use of the carrier in theproduction of such compositions.

In the present invention, pulmonary fibrosis includes not only pulmonaryfibrosis in a restricted sense, but also pulmonary fibrosis in a broadsense that includes co-existence of interstitial pneumonia. Thepulmonary fibrosis of the present invention can be interstitialpneumonia associated with viral pneumonia, fungal pneumonia, mycoplasmalpneumonia, etc., interstitial pneumonia associated with collagen diseasesuch as rheumatoid arthritis, systemic scleroderma, dermatomyositis,polymyositis, mixed connective-tissues disease (MCTD), etc.,interstitial pneumonia associated with radiation exposure, drug-inducedinterstitial pneumonia caused by anticancer agents such as bleomycin,herbal medicines such as Sho-sai-ko-to, interferon, antibiotics,paraquat, etc., and idiopathic interstitial pneumonia such as idiopathicpulmonary fibrosis, nonspecific interstitial pneumonia, acuteinterstitial pneumonia, cryptogenic organizing pneumonia, respiratorybronchiolitis-associated interstitial lung disease, desquamativeinterstitial pneumonia, lymphoid interstitial pneumonia, etc., andaccordingly, it also includes chronic states of such interstitialpneumonia. The pulmonary fibrosis of the present invention preferablyincludes chronic states of drug-induced interstitial pneumonia andidiopathic interstitial pneumonia.

In the composition of the present invention, as long as the retinoidcontained in the carrier is present in a mode that functions as atargeting agent, the carrier may contain a substance to be deliveredwithin its interior, may be attached to the exterior of a substance tobe delivered, or may be mixed with a substance to be delivered.Therefore, depending on the administration route and the manner in whichthe drug is released, etc., the composition may be covered with anappropriate material such as, for example, an enteric coating or amaterial that disintegrates over time, or may be incorporated into anappropriate drug release system.

The composition of the present invention may be administered via variousroutes including both oral and parenteral routes, and examples thereofinclude, but are not limited to, oral, intravenous, intramuscular,subcutaneous, local, intrapulmonary, intra-airway, intratracheal,intrabronchial, nasal, rectal, intraarterial, intraportal,intraventricular, intramedullar, intra-lymph-node, intralymphatic,intrabrain, intrathecal, intracerebroventricular, transmucosal,percutaneous, intranasal, intraperitoneal, and intrauterine routes, andit may be formulated into a dosage form suitable for each administrationroute. Such a dosage form and formulation method may be selected asappropriate from any known dosage forms and methods (see e.g. HyojunYakuzaigaku (Standard Pharmaceutics), Ed. by Yoshiteru Watanabe et al.,Nankodo, 2003).

Examples of dosage forms suitable for oral administration include, butare not limited to, powder, granule, tablet, capsule, liquid,suspension, emulsion, gel, and syrup, and examples of the dosage formsuitable for parenteral administration include injections such as aninjectable solution, an injectable suspension, an injectable emulsion,and a ready-to-use injection. Formulations for parenteral administrationmay be a form such as an aqueous or nonaqueous isotonic sterile solutionor suspension.

The carrier or the composition of the present invention may be suppliedin any form, but from the viewpoint of storage stability, it ispreferably provided in a ready-to-use form, for example in a form thatallows a doctor and/or a pharmacist, a nurse, another paramedic, etc. toprepare it at the place of treatment or in the vicinity thereof. In thiscase, the carrier or the composition of the present invention isprovided as one or more containers containing at least one essentialconstituent element therefor, and it is prepared prior to use, forexample, within 24 hours prior to use, immediately prior to use. Whenpreparing, a reagent, a solvent, preparation equipment, etc. that arenormally available in a place of preparation may be used as appropriate.

The present invention therefore also relates to a preparation kit forthe carrier or the composition, the kit including one or more containerscontaining singly or in combination a retinoid, and/or a substance to bedelivered, and/or a carrier-constituting substance other than theretinoid, and also to a constituent element necessary for the carrier orthe composition provided in the form of such a kit. The kit of thepresent invention may contain, in addition to the above, instructions,an electronic recording medium such as a CD or DVD related to a processfor preparing the carrier and the composition of the present invention,or an administration method, etc. Furthermore, the kit of the presentinvention may include all of the constituent elements for completing thecarrier or the composition of the present invention, but need not alwaysinclude all of the constituent elements. Therefore, the kit of thepresent invention need not include a reagent or a solvent that isnormally available at a place of medical treatment, an experimentalfacility, etc. such as, for example, sterile water, physiologicalsaline, or a glucose solution.

The present invention further relates to a method for controlling theactivity or growth of an extracellular matrix-producing cell in thelung, or a method for treating pulmonary fibrosis, the method includingadministering an effective amount of the composition to a subject inneed thereof. The effective amount referred to here is, in a method fortreating pulmonary fibrosis, pulmonary fibrosis, alleviates itssymptoms, or delays or stops its progression, and is preferably anamount that prevents the onset or recurrence of pulmonary fibrosis orcures it. It is also preferably an amount that does not cause an adverseeffect that exceeds the benefit from administration. Such an amount maybe determined as appropriate by an in vitro test using cultured cells orby a test in a model animal such as a mouse, a rat, a dog, or a pig, andsuch test methods are well known to a person skilled in the art.Moreover, the dose of the retinoid contained in the carrier and the doseof the drug used in the method of the present invention are known to aperson skilled in the art, or maybe determined as appropriate by theabove-mentioned test, etc.

In the method of the present invention, the specific dose of thecomposition administered may be determined while taking intoconsideration various conditions with respect to a subject in need ofthe treatment, such as the severity of symptoms, general healthcondition of the subject, age, body weight, gender of the subject, diet,the timing and frequency of administration, a medicine used incombination, response to the treatment, compliance with the treatment,etc.

As the administration route, there are various routes including bothoral and parenteral routes, and examples thereof include oral,intravenous, intramuscular, subcutaneous, local, intrapulmonary,intra-airway, intratracheal, intrabronchial, nasal, rectal,intraarterial, intraportal, intraventricular, intramedullar,intra-lymph-node, intralymphatic, intrabrain, intrathecal,intracerebroventricular, transmucosal, percutaneous, intranasal,intraperitoneal, and intrauterine routes.

The frequency of administration depends on the properties of thecomposition used and the above-mentioned conditions of the subject, andmay be a plurality of times per day (that is, 2, 3, 4, 5, or more timesper day), once a day, every few days (that is, every 2, 3, 4, 5, 6, or 7days, etc.), a few times per week (e.g. 2, 3, 4 times, etc. per week),every other week, or every few weeks (that is, every 2, 3, 4 weeks,etc.).

In the method of the present invention, the term “subject” means anyliving individual, preferably an animal, more preferably a mammal, andyet more preferably a human individual. In the present invention, thesubject may be healthy or affected by some disorder, and when treatmentof pulmonary fibrosis is intended, it typically means a subject affectedby interstitial pneumonia or pulmonary fibrosis, or having a risk ofbeing affected by them. For example, when prevention of pulmonaryfibrosis is intended, typical examples include, but are not limited to,a subject affected by interstitial pneumonia, in particular byidiopathic interstitial pneumonia.

Furthermore, the term “treatment” includes all types of medicallyacceptable preventive and/or therapeutic intervention for the purpose ofthe cure, temporary remission, or prevention of a disorder, etc. Forexample, the term “treatment” includes medically acceptable interventionof various purposes, including delaying or stopping the progression ofpulmonary fibrosis, regression or disappearance of lesions, preventionof onset and prevention of recurrence of pulmonary fibrosis.

The present invention also relates to a method for delivering a drug toan extracellular matrix-producing cell in the lung, utilizing the abovecarrier. This method includes, but is not limited the carrier, and astep of administering or adding the carrier having the substance to bedelivered carried thereon to a living being or a medium, for example aculture medium, containing an extracellular matrix-producing cell in thelung. These steps may be achieved as appropriate in accordance with anyknown method or a method described In the present specification, etc.The above delivery method may be combined with another delivery method,for example, another delivery method targeted at the lung. Moreover, theabove method includes a mode carried out in vitro and a mode in which anextracellular matrix-producing cell in the lung inside the body istargeted.

EXAMPLES

The present invention is explained in detail by reference to specificexamples below, but these specific examples are for illustrativepurposes, and do not limit the scope of the present invention.

Example 1 Preparation of siRNA

Three types of siRNA targeted at gp46 (GenBank Accession No. M6924 6),which is a rat homologue of human HSP47, and a random siRNA control werepurchased from Hokkaido System Science Co., Ltd. Each siRNA consists of27 bases overhanging on the 3′ side, and the sequences are as follows.

Sequence A: (sense, SEQ ID NO: 1) 5′-GUUCCACCAUAAGAUGGUAGACAACAG-3,(antisense, SEQ ID NO: 2) 5′-GUUGUCUACCAUCUUAUGGUGGAACAU-3, Sequence B:(sense, SEQ ID NO: 3) 5′-CCACAAGUUUUAUAUCCAAUCUAGCAG-3,(antisense, SEQ ID NO: 4)5) 5′-GCUAGAUUGGAUAUAAAACUUGUGGAU-3,(antisense, SEQ ID NO: 6) 5′-UGUCAAUGUAAUGUAAUGGCUCUAGAU-3,Random siRNA: (sense, SEQ ID NO: 7) 5′-CGAUUCGCUAGACCGGCUUCAUUGCAG-3′(antisense, SEQ ID NO: 8) 5′-GCAAUGAAGCCGGUCUAGCGAAUCGAU-3, 

Furthermore, siRNA that was labeled on the 5′ side with the fluorescentdye 6′-carboxyfluorescein (6-FAM) was also prepared.

Example 2 Preparation of siRNA-Containing VA-Bound Liposome

As a liposome, a cationic liposome containing DC-6-14, cholesterol, andDOPE at a molar ratio of 4:3:3 (Lipotrust, Hokkaido System Science Co.,Ltd.) was used. 10 nmol of liposome and 20 nmol of vitamin A (VA:all-trans retinol, Sigma) were mixed in DMSO using a 1.5-mL tube, thendissolved in chloroform, evaporated once, and then suspended in PBS(phosphate buffered saline). Subsequently, the siRNA (10 μg/mL) obtainedin Example 1 and the liposome suspension were mixed at a ratio of 1:1(w/w). Free VA and siRNA contained In the liposome suspension thusobtained were removed by a micropartition system(SartorionVIVASPIN5000MWCOPES), thus giving an siRNA-containing VA-boundliposome (VA-lip-siRNA). The amount of VA added and the amount of VAcontained in the purified liposome were measured by HPLC, and theproportion of VA bound to the liposome was examined; as a result, it wasfound that the majority of the VA (95.6±0.42%) was bound to theliposome. Furthermore, the efficiency of uptake of siRNA into theliposome was measured by RiboGreen assay (Molecular Probes), and it wasas high as 94.4 ±3.0%. Here, in this formulation, VA was at leastpartially exposed on the surface of the formulation.

Example 3 In Vivo Anti-Pulmonary-Fibrosis Activity of siRNA-ContainingVA-Bound Liposome

(1) Induction of Pulmonary Fibrosis and Administration of Drug

Male S-D rats (6 rats/group, 4 weeks old, Charles River LaboratoriesJapan, Inc.) were administered once with 0.5 mg bleomycin (BLM)dissolved in 0.5 cc of physiological saline into the lungintratracheally by intratracheal cannulation under anesthesia, toproduce a bleomycin pulmonary fibrosis model. With this method, asignificant fibrosis occurs in the lung generally after approximately 3weeks. The VA-lip-siRNA prepared in Example 2 (0.75 mg/kg as an amountof siRNA, 1 ml/kg in volume, i.e., 200 μl for a rat of 200 g) or PBS (1ml/kg in volume) was administered to the rats via the tail vein,starting from the day of administration of bleomycin, at a frequency of3 times/week. The rats were sacrificed 21 days after the bleomycinadministration, and bronchoalveolar lavage (BAL) fluid was analyzed,hydroxyproline in the lung was quantified, and histologicalinvestigation of the lung tissue was performed (see FIG. 1). Student'st-test was used for the evaluation of statistically-significantdifference.

(2) Analysis of BAL Fluid

Analysis of BAL was performed as follows. The rats wereintraperitoneally administered with a lethal dose of pentobarbitalsodium, their thorax was opened, then the trachea was exposed and acannula was inserted into the trachea. Subsequently, 7 ml ofphysiological saline was injected into the lung via the tracheal cannulaand the lavage fluid was collected. This process of injection andcollection was repeated 5 times, and the collected lavage fluids werecombined and centrifuged at 250×g for 10 min. The total number of cellswas counted using a cytometer, and cell fraction count was performedusing a May-Giemsa stained cytospin smear preparation. At least 200cells were counted and classified into macrophage, eosinophil,neutrophil, and lymphocyte in accordance with general morphologicalcriteria. Results of the total cell number count were shown in FIG. 2.This figure shows that the number of cells in the BAL fluid of theVA-lip-siRNA administration group (BLM siRNA) significantly decreased tothe level similar to that of the normal control rat administered withPBS instead of bleomycin, compared to the PBS administration group (BLMalone); suggesting that inflammation has been improved.

(3) Quantification of Hydroxyproline in Lung Tissue

The lung was removed from the rats after BAL, then one whole lung washomogenized using a polytron homogenizer, and the lung hydroxyprolinewas quantified using a method of Kivirikko, et al. (Kivirikko K I, etal. Analytical Biochemistry 1967; 19: 249-255). Namely, the lung tissuewas homogenized in 6-N hydrochloric acid at 110° C. for 18 hr, and 25-μlaliquot was dried at 60° C. It was then dissolved in 1.2 ml of 50%isopropanol, incubated with acetate citrate, pH 6.0, and 200 ml of 0.56%chloramine-T solution at room temperature for 10 min, followed by anincubation at 50° C. for 90 min after the addition of 1 ml of Ehrilichsolution; then absorbance at 560 nm was measured. Results shown in FIG.3 indicate that the amount of lung hydroxyproline (μg) of theVA-lip-siRNA administration group (BLM siRNA) significantly decreasedcompared to that of the PBS administration group (BLM alone), suggestingthat the fibrosis of the lung was significantly suppressed.

(4) Histological Investigation

A part of the removed lung was formalin-fixed in accordance staining,azanstaining (azocarmine, anilineblueorangeGsolution), or immunostainingwith anti-αSMA antibody. Regarding the immunostaining, afterdeparaffinization, samples were reacted with a mouse anti-αSMA antibody(Nichirei Corporation, clone 1A4) as a primary antibody, then with aperoxidase-labeled anti-mouse IgG as a secondary antibody, and developedwith DAB. As shown by the results of HE staining in FIG. 4, in the PBSadministration group (BLM day 21), findings characteristic to pulmonaryfibrosis such as disappearance of pulmonary alveoli, bleeding images andinterstitial hyperplasia were observed, whereas in the VA-lip-siRNAadministration group (BLM+siRNA), fibrotic lesions were significantlyimproved. Similarly, as shown by the results of the azan staining inFIG. 5, in the PBS administration group (BLM alone), a noticeablefibrotic image characterized by enlargement of interstice due to a largequantity of blue-stained collagenous fibrils was observed, whereas inthe VA-lip-siRNA administration group (BLM+siRNA), fibrosis wereapparently suppressed. Moreover, as shown by the results of the αSMAstaining in FIG. 6, while a large number of αSMA-positive cells having abrown color were observed in the interstice in the PBS administrationgroup (BLM alone), the number of αSMA-positive cells significantlydecreased in the VA-lip-siRNA administration group (BLM+siRNA).

Considering that siRNA basically acts in the cytoplasm, the aboveresults indicate that the retinoid functioned as a targeting agent toextracellular matrix-producing cells in the lung, so that a drug isefficiently delivered to these cells, leading to a significantimprovement in disease states of pulmonary fibrosis.

What is claimed is:
 1. A substance delivery carrier comprising aretinoid as a targeting agent, wherein the carrier delivers to anextracellular matrix-producing cell in the lung.
 2. The carrieraccording to claim 1, wherein the retinoid comprises retinol.
 3. Thecarrier according to claim 1, wherein the retinoid content is 0.2-20 wt% of the carrier.
 4. The carrier according to claim 1, wherein thecarrier has a form of a liposome, and the molar ratio of the retinoid tothe lipid contained in the liposome is 8:1-1:4.
 5. A compositioncomprising the carrier according to claim 1 and a drug that controls theactivity or growth of the extracellular matrix-producing cell in thelung.
 6. The composition according to claim 5, wherein the drug isselected from the group consisting of: (i) an agent for inhibitingactivity or production of a bioactive substance selected from the groupconsisting of (1) gelatinase A, (2) gelatinase B and (3)angiotensinogen, (ii) an inhibitor of cell activity, (iii) a growthinhibitor, (iv) an apoptosis-inducing agent, (v) an agent which targetsat least one of extracellular matrix constituent molecules or moleculesinvolved in the production or secretion of said extracellular matrixconstituent molecules, selected from the group consisting of (1) ansiRNA, (2) a ribozyme, (3) an anti-sense nucleic acid, and (4) a DNA/RNAchimeric polynucleotide, and (vi) a vector that expresses said siRNA,said ribozyme, said anti-sense nucleic acid, and/or said DNA/RNAchimeric polynucleotide.
 7. The composition according to claim 6,wherein the molecules involved in the production or secretion of theextracellular matrix constituent molecules is HSP47.
 8. The compositionaccording to claim 5, wherein the drug and the carrier are mixed at aplace of medical treatment or in its vicinity.
 9. A kit for preparingthe composition according to claim 5, wherein the kit comprises one ormore containers comprising singly or in combination the drug forinhibiting activity or growth of extracellular matrix-producing cells inthe lung, and the retinoid.